1. Field of the Invention
This invention relates to the preparation of samples for mass analysis. More particularly, this invention is directed to the preparation of samples of large nonvolatile or thermally labile molecules for mass analysis by freezing a solution of the large molecules, desorbing the frozen molecules, and then, if the desorbed molecules are not already ionized by the desorbing means, ionizing the desorbed molecules prior to introducing the ionized molecules into a mass analysis zone. This method is amenable to mechanical interfacing to liquid chromatography.
2. Description of the Related Art
In the mass analysis of molecules combined with liquid chromatography techniques, a liquid is often vaporized and the molecules in the vapor are ionized prior to introduction into a mass analysis instrument or the solvent is evaporated and the resultant molecules are ionized by desorbing particles or photons for the mass analysis instrument. However, the vaporization or desorption techniques conventionally utilized in preparing samples of large nonvolatile or thermally labile molecules for analysis can result in uncontrolled fragmentation or clustering of such large molecules as well as unsuccessful vaporization or desorption of the solvated molecules (nonreliability).
McLafferty et al U.S. Pat. 3,997,298 describe a liquid chromatography-mass spectrometry apparatus in which a portion (or portions) of the eluted effluent from the column is introduced directly into the ionization chamber of a chemical ionization mass spectrometer with sufficient vacuum pumping.
Brunnee et al U.S. Pat. 4,259,572 describe a method for preparing ionized samples for mass spectral analysis by placing the effluent from a liquid chromatography column on a moving belt conveyor, evaporating the solution medium to leave the substance behind, then moving the belt into the vacuum environment of an ionization chamber associated with a mass spectrometer where it is exposed to highly concentrated energy in the form of ions, electrons, or photons, with the latter coming from a laser. The patentees state that the primary concept common to the various embodiments disclosed requires the exposure of the sample on the conveyor belt always within very short intervals, namely within fractions of seconds, to highly concentrated energy in such a way that by the concentration of energy, the connections between molecules in the sample are locally separated without or prior to any chemical decomposition of the molecules.
Stuke U.S. Pat. 4,686,366 discloses a laser mass spectrometer in which a pulsed laser beam is used to ionize the molecule to be analyzed in the mass spectrometer. The duration of the laser pulse is limited to a few picoseconds to avoid fragmentation of the molecule being analyzed.
While these patents describe methods which purport to avoid undesirable fragmentation of the molecule, in actual practice such techniques are only partially successful, depending upon the particular molecule or molecules being analyzed with some molecules fragmenting in uncontrolled fashion or clustering, and thus such methods are considered to be unpredictable and therefore unreliable.
Becker et al U.S. Pat. 4,733,073, by the inventor and others and assigned to the assignee of this invention, and cross-reference to which is hereby made, teaches a method and apparatus for the spectral analysis of a sample in which a probe beam such as an ion beam, electron beam, or a laser beam, is directed against a surface to be analyzed to cause a sample of material to be removed from the surface. A beam of electromagnetic radiation, such an an untuned high-intensity laser beam, is then directed to a spatial region above the surface causing non-resonant photoionization of the removed surface sample within the beam of radiation. The ionized sample is then subjected to mass spectral analysis.
Over about the last decade, many examples have appeared in the literature describing the mass spectrometry of large nonvolatile or thermally labile molecules by the use of particle or photon bombardment of the sample such as referred to in the patent of Brunnee et al. One article making a review of the development is by K. L. Busch and R. G. Cooks, entitled "Mass Spectrometry of Large, Fragile, and Involatile Molecules", published in Science, Volume 218, (1982) at pages 247-254.
One of the methods described in this article is called fast atom bombardment (FAB). The FAB approach is reviewed in more detail in an article by K. L. Rhinehart, Jr., entitled "Fast Atom Bombardment Mass Spectrometry", published in Science, Volume 218, (1982) at pages 254-260. In the FAB approach, the sample is placed in solution in a liquid of low volatility, commonly glycerol. That solution is then bombarded by atoms of kiloelectron volt energy to directly produce ions for mass analysis. One of the primary potential benefits of the FAB technique is the continual supply of sample molecules to the surface. However, drawbacks include molecular types which avoid segregation to the surface, the accumulation of fragmented molecules at the surface, interferences from the solvent molecules, as well as the resultant difficulty of quantifying relative amounts in mixed samples.
Another mass spectrometric approach involving a solution of the sample involves methods of spraying the solution directly for vaporization and ionization preceding mass analysis. The first of such spraying methods, the so called thermospray technique, is described by M. L. Vestal in an article entitled "High-Performance Liquid Chromatography-Mass Spectrometry", published in Science, Volume 226, (1984) at pages 275-281 and is defined therein as the production of a supersonic jet of vapor with entrained particles or droplets by application of sufficient heat to a capillary to effect controlled partial vaporization of a liquid as it passes through the capillary.
The second spray method, the so called electrospray method, is described by Wong et al in an article entitled "Multiple Charging in Electrospray Ionization of Poly(ethylene glycols)", published in the Journal of Physical Chemistry, Volume 92 (1988) at pages 546-550, in which the authors describe a method wherein the solution enters the electrospray chamber through a metal hypodermic needle which is maintained at a high voltage relative to a metallized capillary tube at ground potential. The high field at the needle tip charges the surfaces of the emerging liquid dispersing it into a fine spray of charged droplets. As the droplets proceed toward the capillary tube, the solvent evaporates leaving only the charged solute which then proceeds through the capillary tube to the mass spectrometer.
However, in each of the above cases, the preparation of large nonvolatile molecules, such as biomolecules, for mass spectral analysis still incurs the risk of uncharacteristic fragmentation or clustering or otherwise uncharacteristic or unreliable isolation of the large molecule while attempting to vaporize the molecule prior to or during ionization of the molecule.